LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

CD44 is associated with a high risk of metastasis, recurrence, and drug resistance in various cancers. Here we report that platelet endothelial aggregation receptor 1 (PEAR1) is a CD44 chaperone protein that protected CD44 from endocytosis-mediated degradation and enhances cleavage of the CD44 intracellular domain (CD44-ICD). Furthermore, we found that lysyl oxidase–like protein 2 (LOXL2), an endogenous ligand of PEAR1, bound to the PEAR1-EMI domain and facilitated the interaction between PEAR1 and CD44 by inducing PEAR1 Ser891 phosphorylation in a manner that was independent of its enzyme activity. Levels of PEAR1 protein and PEAR1 phosphorylation at Ser891 were increased in patients with triple-negative breast cancer (TNBC), were positively correlated with expression of LOXL2 and CD44, and were negatively correlated with overall survival. The level of PEAR1 Ser891 phosphorylation was identified as the best independent prognostic factor in TNBC patients. The prognostic efficacy of the combination of PEAR1 phosphorylation at Ser891 and CD44 expression was superior to that of PEAR1 phosphorylation at Ser891 alone. Blocking the interaction between LOXL2 and PEAR1 with monoclonal antibodies significantly inhibited TNBC metastasis, representing a promising therapeutic strategy for TNBC.


Supplemental Figure 2. PEAR1 exacerbates human TNBC cell metastasis.
(A-C) Stable shPEAR1 and oe-PEAR1 MDA-MB-231 and SUM159 cells, and oe-PEAR1 MDA-MB-468 cells were generated using lentivirus infection.Nontargeting shRNA was used as a negative control for shPEAR1 (shnc).The corresponding empty vector (Vector) was used as a negative control for oe-PEAR1.The expression levels of PEAR1 were confirmed with RT-qPCR and western blotting.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA for qPCR (n = 3; mean ± SD). α-Tubulin, GAPDH, or β-actin was used as an internal control for WB.Representative images of invasive shPEAR1-and oe-PEAR1-treated MDA-MB-231 and SUM159 cells, and oe-PEAR1-treated MDA-MB-468 cells in the transwell assay.Scale bar: 100 μm.Quantification of the proliferation levels of shPEAR1-and oe-PEAR1-treated MDA-MB-231 cells in the BeyoClick™ EdU assay (with TMB) (n = 3; mean ± SEM).(D) ShPEAR1 and shnc MDA-MB-231 cells were injected into the orthotopic fat pads of nude mice and observed for 96 days, after which the growth curves of the tumors in situ were recorded (n = 5 mice per group; mean ± SEM).(E) Representative images of metastatic foci in the lungs and livers of nude mice intravenously injected with shPEAR1 and shnc MDA-MB-231 cells by H&E staining (n = 5 mice per group).Scale bar: 50 μm.Unpaired two-tailed t tests were used in (A), (B), (C), and (D); one-way ANOVA followed by Dunnett's test was used in (A) and (B).***P < 0.001, ****P < 0.0001.

Supplemental Figure 3. PEAR1 exacerbates murine TNBC cell metastasis.
(A) Stable shPear1 4T1 cells were generated using lentivirus infection and confirmed with RT-qPCR and western blotting.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA for qPCR (n = 3; mean ± SD). α-Tubulin was used as an internal control for WB.(B) Representative images of invasive shPear1-treated 4T1 cells in a transwell assay.Scale bar: 100 μm.(C) Quantification of invasive shPear1-treated 4T1 cells in a transwell assay, and quantification of the relative migration area of shPear1-treated 4T1 cells in the wound healing assay, and the viability of shPear1-treated 4T1 cells was assessed with a CCK8 assay kit (n = 3; mean ± SEM).(D) ShPear1 and shnc 4T1 cells were injected into the orthotopic fat pads of BALB/c mice and observed for 20 days, after which the growth curves of the tumors in situ were recorded (n = 5 mice per group; mean ± SEM).Representative images and quantification of metastatic foci in the lungs and livers by H&E staining.Scale bar: 50 μm.Unpaired two-tailed t tests were used in (D); one-way ANOVA followed by Dunnett's test was used in (A) and (C).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplemental Figure 4. CD44-ICD is an important messenger of CD44-mediated intracellular signals in MDA-MB-231 cells.
(A) Representative images of shPEAR1 and oe-PEAR1-treated MDA-MB-231 cells in the mammosphere formation assay.Scale bar: 50 μm.(B) The expression levels of CD44 in total lysates and of CD44-ICD in nuclear proteins of shPEAR1 and oe-PEAR1 SUM159 cells determined by western blotting analysis.GAPDH was used as an internal control for total lysates; histone H3 was used as an internal control for nuclear proteins.(C) CD44 mRNA expression levels in shPEAR1 and oe-PEAR1 SUM159 cells were detected by RT-qPCR.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA (n = 3; mean ± SD). (D) The expression levels of CD44-ICD in nuclear proteins of MDA-MB-231 cells treated with 0, 10, 20, 50, or 100 μM DAPT (an inhibitor of γ-secretase) for 24 h.Histone H3 was used as an internal control for nuclear proteins.(G) The viability curves of MDA-MB-231 cells treated with 0, 50, or 100 μM DAPT were generated with a CCK8 assay kit (n = 3; mean ± SEM).One-way ANOVA followed by Dunnett's test was used in (C), (E), (F), and (G); unpaired two-tailed t tests were used in (C).*P < 0.05, ***P < 0.001, ****P < 0.0001.The western blotting results were representative of three independent experiments.

Supplemental Figure 5. PEAR1 regulates TNBC cell metastasis through CD44.
(A) Stable oe-PEAR1 MCF7 cells were generated using lentivirus infection, and confirmed with RT-qPCR and western blotting.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA for qPCR (n = 3; mean ± SD). α-Tubulin was used as an internal control for WB.(B-D) Representative images and quantification of invasive cells in transwell assay, the relative migration area in wound healing assay, and the viability in a CCK8 assay kit of oe-PEAR1-treated MCF7 cells (n = 3; mean ± SEM).Scale bar: 100 μm.(E) The expression levels of PEAR1 and CD44 in MD-MB-231 cells and MCF7 cells were detected by RT-qPCR and western blotting.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA for qPCR (n = 3; mean ± SD). α-Tubulin was used as an internal control for WB.(F) Stable CD44 knockdown (shCD44) and wide-type (WT) MDA-MB-231 cells, and stable shCD44 and oe-PEAR1 MDA-MB-231 cells were generated using lentivirus infection, and confirmed with RT-qPCR and western blotting.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA for qPCR (n = 3; mean ± SD).GAPDH was used as an internal control for WB.(G-I) The relative migration area in wound healing assay, representative images and quantification of invasive cells in transwell assay, and the viability in a CCK8 assay kit of shCD44-treated MDA-MB-231 cells (WT/oe-PEAR1-treated) cells (n = 3; mean ± SEM).Scale bar: 100 μm.One-way ANOVA followed by Dunnett's test was used in (F), (G), (H), and (I); unpaired two-tailed t tests were used in (A), (B), (C), (D), (E), (G), (H), and (I).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.(G) MS analysis of WNK1-specific peptide (VAIPEVK) from co-IP samples (PEAR1-Flag-interacting proteins) of whole-cell lysates of oe-PEAR1-Flag-treated MDA-MB-231 cells using anti-Flag agarose beads.Cells transfected with the corresponding empty vector plasmids were used as negative controls.(H) Co-IP of whole-cell lysates of oe-PEAR1-Flag-treated MDA-MB-231 cells with anti-Flag agarose beads.Cells transfected with the corresponding empty vector plasmids were used as negative controls.(I-L) PEAR1 phosphorylation was detected with western blotting, representative images and quantification of invasive cells in transwell assay, quantification of the relative migration area in wound healing assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells treated with 0, 5, 10, or 20 μM WNK-IN-11 in a transwell assay (n = 3; mean ± SEM).Scale bar: 100 μm.One-way ANOVA followed by Dunnett's test was used in (B), (E), (J), (K), and (L).**P < 0.01, ***P < 0.001, ****P < 0.0001.The western blotting results were representative of three independent experiments.(E) The interaction between PEAR1 fab-HSA and various PEAR1-ECD domain peptides, PEAR1-EMI domain protein was detected using ELISA (n = 2; mean ± SD). (F) The interaction between LOXL2 and PEAR1-ECD protein competitively blocked by 20 μg/mL PEAR1 fab-HSA was detected using ELISA (n = 2; mean ± SD).

Supplemental
(G-H) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit MDA-MB-231 cells treated with 10 ng/mL LOXL2 and 5 or 10 μg/mL PEAR1 fab-HSA (n = 3; mean ± SEM).Scale bar: 100 μm.(I) Representative images of metastatic foci in the lungs and livers of NOD-SCID mice intravenously injected with MDA-MB-231 cells and treated using PEAR1 fab-HSA at a dose of 3.35 mg/kg for 40 days by H&E staining (n = 5 mice per group).Scale bar: 50 μm.One-way ANOVA followed by Dunnett's test was used in (B), (D) and (H).
Supplemental Figure 9.The LOXL2/PEAR1/CD44 axis is upregulated in TNBC and is associated with poor overall survival.(A) The interaction between anti-phospho-PEAR1 (Ser891) antibody (p-PEAR1-S891 Ab) and p-PEAR1-S891 peptide, or the interaction between the corresponding anti-nonphosphorylated PEAR1 antibody (NP-PEAR1 Ab) and NP-PEAR1 peptide was detected using ELISA.The polyclonal antibody against phosphorylated PEAR1 at Ser891 was prepared by immunizing rabbit and purified with a synthetic p-PEAR1-S891 peptide.
(B) The specificity of the anti-p-PEAR1-S891 Ab was evaluated by dot blotting.The loading content of p-PEAR1-S891 peptide and the corresponding NP-PEAR1 peptide was 7 ng.(C) The expression levels of p-PEAR1-S891 and NP-PEAR1 in total lysates of oe-PEAR1-WT-Flag, oe-PEAR1-All SA-Flag and oe-PEAR1-S891A-Flag MDA-MB-231 cells determined by western blotting analysis.α-Tubulin was used as an internal control.(D) Levels of PEAR1 phosphorylation at Ser891 in MDA-MB-231 cells and SUM159 cells treated with 0, 5, 10, 20 ng/mL LOXL2 for 1 h were detected using western blotting after IP with an anti-PEAR1 antibody.(E) Representative IHC staining of the tissue microarray containing TNBC and corresponding adjacent samples with anti-PEAR1, anti-phospho-PEAR1 (Ser891), anti-LOXL2 and anti-CD44 antibodies.Scale bar: 50 μm.(F) Correlation between the expression levels of PEAR1, phospho-PEAR1 (Ser891), LOXL2 and CD44 in TNBC samples (n = 80; pearson correlation analysis).(G) The prognostic effect of the risk score of LOXL2, PEAR1, phospho-PEAR1 (Ser891), CD44 and their combination with each other on TNBC patients, respectively (time-dependent ROC curve analysis).Pearson correlation analysis was used in (F); time-dependent ROC curve analysis was used in (G).The western blotting results were representative of three independent experiments.

Figure 6 .
PEAR1 phosphorylation at Ser891 is crucial for CD44 function.(A) Five phosphorylated serine residues indicated of PEAR1 were identified in MDA-MB-231 cells by IP-MS analysis.(B) CD44 mRNA expression levels in indicated MDA-MB-231 cells were detected by RT-qPCR.The bar diagram shows the relative expression of mRNAs normalized to that of 18S rRNA (n = 3; mean ± SD). (C) Representative images of indicated MDA-MB-231 cells in the mammosphere formation assay.Scale bar: 50 μm.(D-E) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells indicated (n = 3; mean ± SEM).Scale bar: 100 μm.(F) Representative images of metastatic foci in the lungs and livers of nude mice intravenously injected with indicated MDA-MB-231 cells by H&E staining (n = 5 mice per group).Scale bar: 50 μm.

Figure 7 .
LOXL2 triggers PEAR1 phosphorylation.(A) Silver staining of PEAR1-ECD and its interacting proteins pulled down with Dynabeads in the supernatant of MDA-MB-231 cells, in which 15 μg of PEAR1-ECD-his was exogenously added.The arrow pointed to the specific protein bands of PEAR1-ECD interacting proteins compared to the control (PEAR1-ECD-his was not added exogenously).The bands were further to be cut for MS identification.(B) MS analysis of LOXL2-specific peptide (LGQGIGPIHLNEIQCTGNEK) from the specific bands in (A).(C-D) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells treated with 0, 5, or 10 ng/mL LOXL2 (n = 3; mean ± SEM).Scale bar: 100 μm.(E-F) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells treated with 0.2 nM full-length LOXL2 or truncation LOXL2 (n = 3; mean ± SEM).Scale bar: 100 μm.(G-H) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL Simtuzumab (n = 3; mean ± SEM).Scale bar: 100 μm.One-way ANOVA followed by Dunnett's test was used in (D), (F), and (H).Supplemental Figure 8. Blocking the interaction of LOXL2 with PEAR1 inhibits TNBC metastasis.(A-B) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit of MDA-MB-231 cells treated with or without 10 ng/mL LOXL2 and 1 or 5 μg/mL PEAR1-EMI domain protein (n = 3; mean ± SEM).Scale bar: 100 μm.(C-D) Representative images of invasive cells in transwell assay, and viability curves in a CCK8 assay kit MDA-MB-231 cells with oe-PEAR1-EMI domain deficiency (n = 3; mean ± SEM).Scale bar: 100 μm.